Autoimmune polyglandular syndromes (APS) are classified into four main categories, APS1–APS4. APS1 is caused by AIRE gene loss of function mutations, while the genetic background of the other APS remains to be clarified. Here, we investigated the potential association between AIRE gene promoter Single Nucleotide Polymorphisms (SNPs) and susceptibility to APS. We sequenced the AIRE gene promoter of 74 APS patients, also analyzing their clinical and autoantibody profile, and we further conducted molecular modeling studies on the identified SNPs. Overall, we found 6 SNPs (-230Y, -655R, -261M, -380S, -191M, -402S) of the AIRE promoter in patients’ DNA. Interestingly, folding free energy calculations highlighted that all identified SNPs, except for -261M, modify the stability of the nucleic acid structure. A rather similar percentage of APS3 and APS4 patients had polymorphisms in the AIRE promoter. Conversely, there was no association between APS2 and AIRE promoter polymorphisms. Further AIRE promoter SNPs were found in 4 out of 5 patients with APS1 clinical diagnosis that did not harbor AIRE loss of function mutations. We hypothesize that AIRE promoter polymorphisms could contribute to APS predisposition, although this should be validated through genetic screening in larger patient cohorts and in vitro and in vivo functional studies.

Autoimmune polyglandular syndrome (APS) comprises a complex association of autoimmune pathological conditions. APS Type 1 originates from loss-of-function mutations in the autoimmune regulator (AIRE) gene. APS2, APS3 and APS4 are linked to specific HLA alleles within the major histocompatibility complex, with single-nucleotide polymorphisms (SNPs) in non-HLA genes also contributing to disease. In general, variability in the AIRE locus and the presence of heterozygous loss-of-function mutations can impact self-antigen presentation in the thymus. In this study, whole-exome sequencing (WES) was performed on a sixteen-year-old female APS3A/B patient to investigate the genetic basis of her complex phenotype. The analysis identified two variants (p.Arg111Trp and p.Thr101Ile) of the hepatitis A virus cell receptor 2 gene (HAVCR2) encoding for the TIM-3 (T cell immunoglobulin and mucin domain 3) protein. These variants were predicted, through in silico analysis, to impact protein structure and stability, potentially influencing the patient’s autoimmune phenotype. While confocal microscopy analysis revealed no alteration in TIM-3 fluorescence intensity between the PBMCs isolated from the patient and those of a healthy donor, RT-qPCR showed reduced TIM-3 expression in the patient’s unfractionated PBMCs. A screening conducted on a cohort of thirty APS patients indicated that the p.Thr101Ile and p.Arg111Trp mutations were unique to the proband. This study opens the pathway for the search of TIM-3 variants possibly linked to complex autoimmune phenotypes, highlighting the potential of novel variant discovery in contributing to APS classification and diagnosis.

Autoimmune polyglandular syndrome (APS) comprises a complex association of autoimmune pathological conditions. APS Type 1 originates from loss-of-function mutations in the autoimmune regulatory (AIRE) gene. APS2, APS3, and APS4 are linked to specific HLA alleles within the major histocompatibility complex, with single nucleotide polymorphisms (SNPs) in non-HLA genes also contributing to disease. In general, variability in the AIRE locus and the presence of heterozygous loss-of-function mutations can impact self-antigen presentation in the thymus. In this study, Whole Exome Sequencing (WES) was performed on a sixteen-year-old female APS3A/B patient to investigate the genetic basis of her complex phenotype. The analysis identified two variants (p.Arg111Trp and p.Thr101Ile) of the hepatitis A virus cell receptor 2 gene (HAVCR2) encoding for the TIM-3 (T cell immunoglobulin and mucin domain 3) protein. These variants were predicted, through in silico analysis, to impact protein structure and stability, potentially influencing the patient’s autoimmune phenotype. While Confocal Microscopy analysis revealed no alteration in TIM-3 distribution between PBMCs isolated from the patient and those of a healthy donor, RT-qPCR showed reduced TIM-3 expression in the patient’s cells. A screening conducted on a cohort of thirty APS patients indicated that the p.Thr101Ile and p.Arg111Trp mutations were unique for the proband. This study opens the pathway for the search of TIM-3 variants possibly linked to complex autoimmune phenotypes, highlighting the potential of novel variant discovery in contributing to APS classification and diagnosis.

Central tolerance is provided by the autoimmune regulator (AIRE) expressing medullary thymic epithelial cells through high-avidity recognition of self-antigens. Peripheral mechanisms regulate adaptive immunity by deleting autoreactive T-cells that escape thymic selection, or by inducing their functional unresponsiveness through interaction with antigen-presenting cells, exposing cognate antigens. Multiple types of extrathymic AIRE-expressing cells residing in secondary lymphoid organs have been consistently described. We investigated whether AIRE binds to promoters of known autoantigens in human peripheral blood mononuclear cells by chromatin immunoprecipitation from four healthy donors using an anti-AIRE monoclonal antibody. Quantitative real-time PCR was used to detect AIRE occupancy at promoters of selected autoantigens. Accordingly, we revealed promoter amplicons of all tested autoantigen genes, and their corresponding transcripts. Expression of AIRE was further confirmed at both transcriptional and protein levels. Overall, although the role of AIRE in regulating autoantigen expression in thymic epithelial cells has been well reported, our study provides preliminary descriptive evidence of AIRE occupancy at promoters of known autoantigens in human bulk peripheral blood mononuclear cells, suggesting the presence of AIRE-mediated regulatory events in peripheral blood. Our data provide a rationale for future investigations aimed at elucidating the underlying molecular and immunological mechanisms.

Central tolerance is provided by the AIRE-expressing medullary thymic epithelial cells, through high avidity recognition of self-antigens. Nevertheless, peripheral mechanisms regulate adaptive immunity by deleting autoreactive T-cells that escape thymic selection or inducing their functional unresponsiveness. These mechanisms require interaction with antigen presenting cells exposing cognate antigen. As regard multiple types of extrathymic AIRE-expressing cells, residing in secondary lymphoid organs, were described. In this study we aimed to provide evidence for AIRE binding to promoter regions of known autoantigens in human peripheral blood mononuclear cells (PBMC) in an attempt to elucidate whether this non-classical transcriptional factor could play a role in the pe-ripheral expression of self-antigens. Chromatin immunoprecipitation (ChIP) of 4 normal human PBMC samples was performed using anti-AIRE monoclonal antibody. Quantitative-Real-Time PCR (qRT-PCR) was used to detect AIRE-binding at promoters of known autoantigens, including thyroidrelated thyroglobulin, thyroperoxidase, thyrotropin-receptor, Type 1 diabetes-related autoantigens, i.e. insulin and zinc transporter 8, and to confirm their expression in PBMC. ChIP evidenced amplicons of promoter regions of mentioned autoantigens by qRT-PCR. Expression of AIRE and of autoantigens was confirmed in the same human PBMC samples. This study provides the first evidence that AIRE binds promoters of known autoantigens in human PBMC, supporting its expression and potential role in modulating peripheral self-antigen expression.